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Hydrophobic clusters raise the threshold hydrophilicity for insertion of transmembrane sequences in vivo.


Insertion of a nascent membrane protein segment by the translocon channel into the bilayer is naturally promoted by high segmental hydrophobicity, but its selection as a transmembrane (TM) segment is complicated by the diverse environments (aqueous vs. lipidic) the protein encounters, and by the fact that most TM segments contain a substantial amount (~30%) of polar residues as required for protein structural stabilization and/or function. To examine the contributions of these factors systematically, we designed and synthesized a peptide library consisting of pairs of compositionally identical - but sequentially different - peptides with 19-residue core sequences varying (i) in Leu positioning (with five or seven Leu residues clustered into a contiguous 'block' in the middle of the segment, or 'scrambled' throughout the sequence); and (ii) in Ser content (0-6 residues). The library was analyzed by a combination of biophysical and biological techniques, including HPLC retention times, circular dichroism measurements of helicity in micelle and phospholipid bilayer media, and relative blue shifts in Trp fluorescence maxima; and by extent of membrane insertion in a translocon-mediated assay using microsomal membranes from dog pancreas endoplasmic reticulum (ER). We found that local blocks of high hydrophobicity heighten the translocon's propensity to insert moderately hydrophilic sequences, until a "threshold hydrophilicity" is surpassed whereby segments no longer insert even in the presence of Leu blocks. This study codifies the prerequisites of apolar/polar content and residue positioning that define nascent TM segments, illustrates the accuracy in their prediction, and highlights how a single disease-causing mutation can tip the balance toward anomalous translocation/insertion.